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BOVINE RESERVOIR FORVEROTOXIN-PRODUCINGESCHERICHIA COLI O157:H7

SIR,-Verotoxin-producing Escherichia coli (VTEC) have awell-established association with haemorrhagic colitisl.2 and thehaemolytic uraemic syndrome.3,4 Although only limitedinformation is available about the sources and reservoirs of VTECin nature, it is probable that these bacteria are widely distributed inthe intestinal tracts of animals and that food-products of animalorigin are primary sources of human infection. VTEC belonging toseveral 0 serogroups, including human types 26 and 11 l, have beenrecovered from bovine faeces,5,6 and types 26 and 68 have beenisolated from cheese.7 However, little is known about the reservoirsof the best studied VTEC serotype, 0157:H7, which has beenimplicated in several VTEC outbreaks, including two in which thesource was shown to be undercooked hamburger meat.1An opportunity to investigate the reservoir of this organism

presented itself during a large outbreak of VTEC 0157:H7infection among kindergarten children in south-west Ontario. InApril, 1986, 60 children aged 4 to 5 and 14 adults visited a dairyfarm. Afterwards 48 had abdominal cramps and most had waterydiarrhoea; haemolytic uraemic syndrome developed in 3 of thechildren. VTEC 0157:H7 infection was diagnosed in 43 of thesymptomatic cases by isolation of this organism from stools8 and/ordemonstration of free verotoxin in faecal filtrates.3,4 A significantassociation was found between infection and the consumption ofunpasteurised milk. Faecal samples from 67 healthy calves and cows’on the farm were investigated for E coli 0157:H7 on MacConkey-sorbitol medium-,9,11 colonies that were sorbitol negative after

overnight incubation at 36°C were examined for agglutination in0157 antiserum.lO VTEC 0157:H7 was isolated from 1 animal.

Subsequently, 42 samples were re-examined by a sensitive methodthat involves the detection of verotoxin in polymyxin B extracts ofcolony sweeps from primary agar cultures." This procedure, whichcan detect about 1 % VTEC colonies in mixed cultures, resulted inthe isolation of VTEC from an additional 6 animals. One of theseisolates was of serotype 0157:H7; the other 5 have not yet beenserotyped.Our findings provide direct evidence that cattle may be primary

reservoirs of VTEC serotype 0157:H7 bacteria that are associatedwith human disease.

Reference Bacteriology Laboratories,Central Public Health Laboratory,Ontario Ministry of Health A. A. BORCZYK

Department of Bacteriology,Hospital for Sick Children, Toronto M. A. KARMALI

National Enteric Reference Centre,Laboratory Centre for Disease Control,Tunney’s Pasture,Ottawa, Ontario K1A 0L2, Canada H. LIOR

Lambton Health Unit,Sarnia, Ontario L. M. C. DUNCAN

1. Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rareEscherichia coli serotype. N Engl J Med 1983; 308: 681-85.

2. Johnson WM, Lior H, Bezanson GS. Cytotoxic Escherichia coli O157:H7 associatedwith haemorrhagic colitis in Canada. Lancet 1983; i: 76.

3. Karmali MA, Steele BT, Petric M, Lim C. Sporadic cases of haemolytic uraemicsyndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coliin stools. Lancet 1983; i: 619-20.

4. Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. The associationbetween idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 1985; 151: 775-82.

5. Sherwood D, Snodgrass DR, O’Brien AD. Shiga-like toxin production fromEscherichia coli associated with calf diarrhea. Vet Record 1985; 116: 217-18.

6. Mohammad A, Peiris JSM, Wijewanta EA, Mahalingam S, Gunasekara G. Role ofverocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhea. FEMSMicrobiol Letters 1985; 26: 281-83.

7. Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli.Infect Immun 1977; 18: 775-79.

8. Wells JG, Davis BR, Wachsmuth IK, Riley LW, Remis RS, Sokolow R, Moms GK.Laboratory investigation of hemorrhagic colitis outbreaks associated with a rareEscherichia coli serotype. J Clin Microbiol 1983; 18: 512-20.

9. Rappaport F, Henig E. Media for the isolation and differentiation of pathogenic E coliserotypes O111 and O55. J Clin Pathol 1952; 5: 361-62.

10. Farmer JJ III, Davies BR. H7 antiserum-sorbitol fermentation medium: a single tubescreening medium for detecting Escherichia coli O157:H7 associated with

hemorrhagic colitis. J Clin Microbiol 1985; 22: 620-25.11. Karmali MA, Petric M, Corazon L, Cheung R, Arbus GS. Sensitive method for

detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures byuse of colony sweeps and polymyxin extraction of verotoxin. J Clin Microbiol 1985,22: 614-19.

SIGNIFICANCE OF GROUP B STREPTOCOCCI

(STREP AGALACTIAE) IN CHILDREN’S URINARYTRACT INFECTION

SIR,—Miss Collins and her colleagues (Aug 30, p 479) emphasisethe importance of incubating urinary specimens for up to 48 h in7% CO2 to detect fastidious species. They also note that 20% ofStreptococcus agalactiae would not have been detected afterovernight incubation on CLED agar and even more of the fastidiousstrains would have been missed. In 1979 the same laboratoryisolated 82 slow-growing, COz-dependent organisms(Corynebacterium spp, Lactobacillus spp, and Strep millera) frompatients with urethral syndrome.! This was followed by a debate onthe signficance of anaerobic and microaerophilic species as urinarytract pathogens.2-4 Our experience prompts similar questions aboutthe pathogenicity of Strep agalactiae. From December, 1984, toDecember, 1986, 12 children aged 4-16 years produced positiveurine cultures (> 105/ml) for Strep agalactiae (table). 7 were

symptom-free. 9 patients had abnormal renal tracts.

CLINICAL DETAILS OF TWELVE CHILDREN WITH POSITIVE URINE

CULTURES FOR STREP AGALACTIAE

*Dysuria in cases 4, 6, 7, and 9; tiredness and pallor in case 3.tAbnormalines were: extensive bilateral cortical scamng (case 1); posturethral valves,bladder diverticula, unilateral renal hypoplasia (case 2); bilateral vesicoureteric reflux (case3); bilateral vesicoureteric reflux, unilateral reflux nephropathy (case 4); bilateralvesicouretenc reflux, unilateral reflux nephropathy (case 5), bifid pelvis (case 6); spinabifida, obstructive uropathy, continuous bladder drainage (case 8); unilateral hypoplasia(case 10); bilateral reflux nephropathy (case 12).

Patients 3 and 7 were treated on the first positive urine culture,and both symptoms and organisms cleared. Patient 9 was treatedafter a delay of 4 months when a second culture was positive; thisgirl’s symptoms were severe and persistent and were relievedpromptly by co-trimoxazole. In patient 6 symptoms and organismscleared without therapy and in patient 4 these cleared after relief ofurinary retention. Thus 3 patients with symptoms and 7

symptomless patients received no therapy.We used ’Uricult’ (Orion, Helsinki) dip-slides coated with

MacConkey and CLED (cystine-Iactose-electrolyte-deficient) agarincubated in air at 37°C for 24 h. Strep agalactiae appeared as palepink colonies resembling Enterococcusfaecalis on MacConkey agarbut appeared on CLED agar in only 4 cases; they were fullyidentified using the API 20 STREP system. This accords withCollins et al who would have missed 11/43 at 24 h. Theyrecommend incubating CLED medium in 7% CO2 for 48 h toisolate Strep agalactiae and this confirms the work of Jokipii andJ okipü. All our Strep agalactiae strains were sensitive to ampicillin,amoxycillin, co-trimoxazole, sulphonamide, and cephradine.

Clinicians often ask clinical microbiology laboratories about thepotential pathogenicity of isolates. There is no sharp distinctionbetween pathogen and commensal. With Strep agalactiae, which israrely isolated from urine, it is difficult to be dogmatic in the